A research group of three people, skilled in different aspects of the problem, will investigate the biophysics of protein folding using various carbonic anhydrases as models. The work is already in progress so that important information on folding is available. The major objective of the project is to determine the pathway of folding of the high activity, type II protein that is found in bovine and human erythrocytes. The protein (mol wt 30,000) is about twice as large as most proteins for which there is information on the folding pathway. A unique advantage of carbonic anhydrase for this kind of project, over the usual small proteins that have been investigated (e.g. RNase, lysozyme, cytochrome c), is that the high degree of folding cooperativity that makes the direct observation of kinetic intermediates exceptionaly difficult, is not present in this protein. Well-populated intermediates in the folding of bovine erythrocyte carbonic anhydrase are known. The pathway will be determined by characterizing the structures and kinetics of the folding intermediates. Dynamic NMR, stopped-flow CD, polarization modulated emission, electronic absorbtion spectroscopy, and catalytic activity measurements will be used. Measurements will be made under varying conditions, especially low temperatures, where intermediates are stable relative to the unfolded protein chain. The proposed research is intended to fill a major gap in knowledge on protein chemistry and has exciting significance to the question of the fundamental principles that govern the formation of the three-dimensional structure of a protein from the nascent polypeptide chain. Furthermore, characterization of the folding intermediates can provide a benchmark for theorists working on computational studies of protein folding, structure prediction, and folding simulation. Numerous health-related topics in protein chemistry could be advanced by the new knowledge obtained in the project.